Immunological reagents and diagnostic methods for the detection of human immunodeficiency virus type 2 utilizing multimeric forms of the envelope proteins GP300, P200, and P90/80

ABSTRACT

Four glycoproteins of apparent molecular weights 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) are detectable in human immunodeficiency virus type 2 (HIV-2) infected cells. The gp125 and gp36 are the external and transmembrane components, respectively, of the envelope glycoproteins of HIV-2 mature virions. The gp300, which is a dimeric form of gp14O, the precursor of HIV-2 envelope glycoprotein, is probably formed by a pH dependent fusion in the endoplasmic reticulum. Such a doublet is also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 does not form a dimer during its processing. Experiments carried out with various inhibitors of oligosaccharide trimming enzymes suggest that transient dimerization of the glycoprotein precursor is required for its efficient transport to the Golgi apparatus and for its processing. The gp300 is useful for detecting antibodies to HIV-2 antigens in human body fluids and for raising antibodies to gp300.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 08/002,756, filed Jan. 13, 1993 (pending), which is a division ofapplication Ser. No. 07/356,459, filed May 25, 1989, now U.S. Pat. No.5,208,321. Ser. No. 08/002,756 is also a continuation-in-part ofapplication Serial No. 07/204,346, filed Jun. 9, 1988 (abandoned), whichis a continuation application of Ser. No. 07/804,712, filed Dec. 6,1991, now U.S. Pat. No. 5,312,902. The related applications arespecifically incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to viral proteins and glycoproteins, tocompositions containing these proteins, to methods of preparing theproteins, and to their use in detecting viral infection.

[0003] The etiological agent of acquired immunodeficiency syndrome(AIDS) is the retrovirus referred to as human immunodeficiency virus(HIV) (Montagnier et al., 1984). To date, two related but distinctviruses, HIV-1 and HIV-2, have been identified (Barre-Sinoussi et al.,1983; Popovic et al., 1984; Levy et al., 1984; Wain-Hobson et al.,1985a; Clavel et al., 1986a; Brun-Vezinet et al., 1987; Guyader et al.,1987). HIV-2 is closely related to simian immunodeficiency virus (SIV),which causes an AIDS-like disease in macaques (Daniel et al, 1985;Sonigo et al., 1985; Chakrabarti et al., 1987).

[0004] HIV-1, HIV-2, and SIV show all the features of retrovirus familymembers (Wain-Hobson et al., 1985b; Montagnier and Alizon, 1987; Guyaderet al., 1987; Chakrabarti et al., 1987). Their proviral genomes containtwo long terminal repeats (LTRs) and three essential genes required forvirus replication encoding the viral internal structural proteins (gag),the reverse transcriptase (pol), and the envelope glycoproteins (env) ofthe virus. In addition to these genes, both HIVs and SIV containadditional genes encoding the proteins that regulate viral expression(tat and art/trs) and three other genes encoding proteins of unknownfunction (Q or sor, F or 3′ orf, and R). The only notable difference inthe genetic organizations of HIV-1, HIV-2, and SIV resides in the openreading frame referred to as X, which is absent in HIV-1.

[0005] Alignments of the nucleotide sequences of HIV-1, HIV-2, and SIVreveal a considerable homology between HIV-2 and SIV. These two virusesshare about 75% overall nucleotide sequence homology, but both of themare only distantly related to HIV-1 with about 40% overall homology(Guyader et al., 1987; Chakrabarti et al., 1987). At the protein level,the gag and pol proteins of HIV-1, HIV-2, and SIV are antigenicallycross-reactive, whereas env proteins are cross-reactive only betweenHIV-2 and SIV (Clavel et al., 1986b, 1987).

[0006] HIV-1, HIV-2, and SIV are both tropic and cytopathic for CD4positive T lymphocytes (Dagleish et al., 1984; Klatzman et al., 1984;McDougal et al., 1985; Clavel et al., 1986b, 1987; Kannagi et al., 1985;Fultz et al., 1986). A great number of studies have indicated that CD4functions as the cellular receptor for HIV-1 (for references see Weiss,1988).

[0007] The HIV-1 env gene codes for a 160 Kd glycoprotein that isproteolytically cleaved to yield the extracellular and transmembraneproteins, gp120 and gp41, respectively (Montagnier et al., 1985). It hasbeen demonstrated that HIV-1 recognition of CD4 is mediated by gp120.This complex gp120-CD4 can be identified by co-immunoprecipitation usingantibodies specific for the CD4 antigen (McDougal et al., 1986).Following the binding of gp120 to CD4, the entry of HIV-1 into the cellmight occur by viral envelope cell membrane fusion (Lifson et al., 1986;Sodroski et al., 1986; Stein et al., 1987; McClure et al., 1988). Aputative fusogenic domain in gp41 (Kowalski et al., 1987), which has asequence homologous to other fusion peptides (Phe-Leu-Gly; Gallaher,1987), might provide at least one HIV fusion site necessary for thisprocess (Marsh and Dalgleish, 1988).

[0008] In the case of HIV-2, a high molecular weight protein of about130 Kd to about 140 Kd has been associated with the major envelopeglycoprotein (Clavel et al., Science, 233:343-346, 1986). Anotherglycoprotein having a molecular weight of 120 Kd has been associatedwith the external glycoprotein of HIV-2 (Guyader et al., Nature,362:662-669, 1987). Nevertheless, detailed information for HIV-2envelope proteins and glycoproteins and their cleavage products andprecursors is lacking.

[0009] There exists a need in the art for additional information on thestructure and in vivo processing of HIV-2 proteins, and especially HIV-2envelope proteins and glycoproteins. Such information would aid inidentifying HIV-2 infection in individuals. In addition, such findingscould aid in elucidating the mechanism by which HIV-2 infection andvirus proliferation occur and thereby make it possible to devise modesof intervening in viral processes.

SUMMARY OF THE INVENTION

[0010] This invention aids in fulfilling these needs in the art byproviding HIV-2 envelope proteins and glycoproteins in purified form.Four glycoproteins of apparent molecular weights 300,000, 140,000,125,000, and 36,000 daltons (gp300, gp140, gp125, and gp36) aredetectable in human immunodeficiency virus type 2 (HIV-2) infectedcells. The gp125 and gp36 are the external and transmembrane components,respectively, of the envelope glycoproteins of HIV-2 mature virions. Ithas now been that the gp300 is a dimeric form of gp140, which is theprecursor of HIV-2 envelope glycoprotein. This invention thus providesgp300 glycoprotein of HIV-2 and human retroviral variants of HIV-2 inpurified form.

[0011] This invention also provides proteins of HIV-2 or of a humanretroviral variant of HIV-2 having apparent molecular weights of about200 Kd (p200) and about 90 to about 80 Kd (p90/80). These proteins aresubstantially unglycosylated and are in a purified form.

[0012] A similar high molecular weight glycoprotein of SimianImmunodeficiency Virus (SIV) or of a Simian retroviral variant of SIVhas also been discovered. This glycoprotein is a precursor of anenvelope glycoprotein of SIV and has an apparent molecular weight ofabout 300 Kd (gp300_(SIV)). This glycoprotein is also provided in apurified form.

[0013] This invention also provides labeled gp300 of HIV-2 and gp300 ofSIV. Preferably, the labeled glycoproteins are in purified form. It isalso preferred that the labeled glycoprotein is capable of beingimmunologically recognized by human body fluid containing antibodies toHIV-2 or SIV. The gp300 glycoproteins can be labeled, for example, withan immunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

[0014] Immunological complexes between the proteins and glycoproteins ofthe invention and antibodies recognizing the proteins and glycoproteinsare also provided. The immunological complexes can be labeled with animmunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

[0015] Furthermore, this invention provides a method for detectinginfection of cells by human immunodeficiency virus type-2 (HIV-2). Themethod comprises providing a composition comprising cells suspected ofbeing infected with HIV-2, disrupting cells in the composition to exposeintracellular proteins, and assaying the exposed intracellular proteinsfor the presence of gp300 glycoprotein of HIV-2. The exposedintracellular proteins are typically assayed by electrophoresis or byimmunoassay with antibodies that are immunologically reactive with gp300glycoprotein of HIV-2.

[0016] This invention provides still another method of detectingantigens of HIV-2, which comprises providing a composition suspected ofcontaining antigens of HIV-2, and assaying the composition for thepresence of gp300 glycoprotein of HIV-2. The composition is typicallyfree of cellular debris.

[0017] A method of distinguishing HIV-2 infection from HIV-1 infectionin cells suspected of being infected therewith has also been discovered.The method comprises providing an extract containing intracellularproteins of the cells, and assaying the extract for the presence ofgp300 glycoprotein. The gp300 is characteristic of HIV-2, but theglycoprotein has not been found in extracts of HIV-1 cell cultures.

[0018] In addition, this invention provides a method of making gp300glycoprotein of HIV-2, which comprises providing a compositioncontaining cells in which HIV-2 is capable of replicating, infecting thecells with HIV-2, and culturing the cells under conditions to causeHIV-2 to proliferate. The cells are then disrupted to exposeintracellular proteins. The gp300 glycoprotein is recovered from theresulting exposed intracellular proteins.

[0019] This invention also provides an in vitro diagnostic method forthe detection of the presence or absence of antibodies which bind to anantigen comprising the proteins or glycoproteins of the invention ormixtures of the proteins and glycoproteins. The method comprisescontacting the antigen with a biological fluid for a time and underconditions sufficient for the antigen and antibodies in the biologicalfluid to form an antigen-antibody complex, and then detecting theformation of the complex. The detecting step can further comprisemeasuring the formation of the antigen-antibody complex. The formationof the antigen-antibody complex is preferably measured by immunoassaybased on Western Blot technique, ELISA (enzyme linked immunosorbentassay), indirect immunofluorescent assay, or immunoprecipitation assay.

[0020] A diagnostic kit for the detection of the presence or absence ofantibodies, which bind to the proteins or glycoproteins of the inventionor mixtures of the proteins and glycoproteins, contains antigencomprising the proteins, glycoproteins, or mixtures thereof and meansfor detecting the formation of immune complex between the antigen andantibodies. The antigens and the means are present in an amountsufficient to perform the detection.

[0021] Precursors of the envelope glycoproteins of HIV-2 and SIV can beprepared according to this invention. Specifically, this inventionprovides a method of preparing the precursors, which comprises providingan extracellular composition containing gp300 glycoprotein of HIV-2 orSIV at a pH of at least about 6.5. The pH of the composition is thenlowered to a value of about 4 to about 6.0 in order dissociate the gp300glycoprotein into gp140 glycoprotein of HIV-2 or gp140 glycoprotein ofSIV.

[0022] Finally, this invention provides an immunogenic compositioncomprising a protein or glycoprotein of the invention in an amountsufficient to induce an immunogenic response in vivo, in associationwith a pharmaceutically acceptable carrier therefor.

[0023] The proteins and glycoproteins of this invention are thus usefulas a portion of a diagnostic composition for detecting the presence ofantibodies to antigenic proteins associated with HIV-2 and SIV. Inaddition, the proteins and glycoproteins can be used to raise antibodiesfor detecting the presence of antigenic proteins associated with HIV-2and SIV. The proteins and glycoproteins of the invention can be alsoemployed to raise neutralizing antibodies that either inactivate thevirus, reduce the viability of the virus in vivo, or inhibit or preventviral replication. The ability to elicit virus-neutralizing antibodiesis especially important when the proteins and glycoproteins of theinvention are used in vaccinating compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] This invention will be described in greater detail by referringto the drawings in which:

[0025]FIG. 1A is a fluorograph in which high molecular weight proteinsof HIV-1 and HIV-2 are compared after electrophoresis in apolyacrylamide SDS gel;

[0026]FIG. 1B depicts the result of electrophoresis of HIV-2glycoproteins in an acrylamide gel;

[0027]FIG. 2 depicts the result of two dimensional gel electrophoreticanalysis of HIV-2 glycoproteins;

[0028]FIG. 3(a) is a fluorograph of dissociated gp300 of HIV-2;

[0029]FIG. 3(b) and FIG. 3(c) are fluorographs of denatured gp300 ofHIV-2;

[0030]FIG. 4 shows the result of electrophoresis of HIV-2 glycoproteinsafter the glycoproteins were digested with beta-N-acetylglucosaminidaseH (endo H);

[0031]FIG. 5 is a fluorograph of a polyacrylamide gel afterelectrophoresis of HIV-2 glycoproteins, which were isotopically labeledwith ¹⁴C-mannose or ³H fucose;

[0032]FIG. 6 shows the result of electrophoresis of HIV-2 envelopeproteins obtained from cultures in which N-linked glycosylation wasinhibited by the antibiotic tunicamycin;

[0033]FIG. 7 is a fluorograph of a polyacrylamide gel afterelectrophoresis of HIV-2 envelope glycoproteins obtained from cellcultures with and without oligosaccharide trimming inhibitors;

[0034]FIGS. 8A and 8B depict the results of electrophoresis of HIV-2glycoproteins obtained during pulse-chase experiments in HIV-2 infectedCEM cells in the absence (control) or presence of castanospermine (FIG.8A) or monensin (FIG. 8B);

[0035]FIG. 9 is a fluorograph of polyacrylamide gels afterelectrophoresis of SIV envelope glycoproteins labeled with³⁵S-methionine, ³H-fucose, or ¹⁴C-mannose; and

[0036]FIG. 10 is a schematic pathway postulated for in vivo processingof HIV-2 envelope glycoprotein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0037] As a result of this invention, the processing of HIV-2 envelopeglycoproteins has now been characterized. Four glycoproteins referred toas gp300, gp140, gp125, and gp36 are synthesized in HIV-2 infectedcells. The gp125 and gp36 correspond to the external and transmembraneglycoproteins of HIV-2 virion, whereas gp300 and gp140 are onlydetectable in infected cells. The gp300 is a dimeric form of gp140,which is the immature precursor of HIV-2 envelope glycoprotein. Thisdimer is very stable since it resists ionic and non-ionic detergents,high salt, 4 M urea, and reducing agents. However, the dimer can bedissociated in acidic pH to yield gp140.

[0038] Dimerization occurs in the endoplasmic reticulum after theremoval of glucose residues by glucosidases I and II, and after theaction of Golgi mannosidases, the dimer becomes dissociated probably dueto a shift in pH of the environment in trans Golgi. Finally, proteolyticcleavage of the mature precursor occurs outside the Golgi.

[0039] Transient dimer formation of the glycoprotein precursor seems tobe an intrinsic property of the polypeptide moiety of HIV-2 envelope.This is a novelty in the mechanism of glycoprotein processing withN-linked oligosaccharide chains. It is hypothesized that conformationalmodifications brought about by the formation of this dimer are necessaryfor transport of the glycoprotein precursor to the Golgi apparatus.

[0040] I. IDENTIFICATION OF THE HIV-2 ENVELOPE GLYCOPROTEINS

[0041] Recently, it has been reported that the envelope gene of HIV-2(ROD isolate) encodes a precursor glycoprotein that is then cleavedproteolytically to yield a 120 Kd extracellular glycoprotein and a 36 Kdtransmembrane glycoprotein (Clavel et al., 1986a and 1986b). To identifythe precursors of the HIV-2 glycoproteins, viral proteins in infectedcells as well as in virus particles were studied. For comparison, thesynthesis of HIV-1 proteins in cells infected with HIV-1 (BRU isolate)were also studied. The results are shown in FIG. 1 and were obtained asfollows.

[0042] A. Comparison of high molecular weight proteins of HIV-1 andHIV-2 (FIG. 1A)

[0043] CEM cells infected with HIV-1 or HIV-2 were labeled with³⁵S-methionine (200 μCi/ml; 4×10⁶ cells/ml) for 18 hours. Extracts fromthese infected cells (CELL) and their corresponding culture medium (SN)were purified on specific immunoaffinity columns:

[0044] HIV-1 serum-Sepharose specific for HIV-1 proteins (Krust et al.,1988), and

[0045] HIV-2 serum-Sepharose specific for HIV-2 proteins.

[0046] (See “Experimental Procedures”).

[0047] These purified proteins were analyzed by electrophoresis in a7.5% polyacrylamide SDS-gel containing 6 M urea. A fluorograph of thegel is presented in FIG. 1. The sizes of the HIV-1 and HIV-2 proteinsare indicated on the left and right of the lanes shown in FIG. 1.

[0048] Referring to FIG. 1A, the p68 and p55 are the reversetranscriptase and the gag precursor, respectively. The gp160 and gp120are the glycoprotein precursor of HIV-1 envelope and its cleavedproduct.

[0049] Three major high molecular weight glycoproteins of 300, 140, and125 Kd are detectable in HIV-2 infected cells (FIG. 1A). The proteinsare specific to HIV-2 because they are absent in non- infected cells andbecause they could be consistently identified by all HIV-2, but notHIV-1, seropositive sera in an immunoprecipitation assay (data notshown).

[0050] In side by side comparison, the electrophoretic mobility of thesethree HIV-2 proteins is clearly different from that of the 160 Kd HIV-1precursor glycoprotein (gp160) and one of its cleaved products, 120 Kdexternal envelope glycoprotein (gp120; FIG. 1A). It should be noted thatthe resolution of the 140 and 125 Kd proteins of HIV-2 from one anothercan be clearly observed in polyacrylamide-SDS gels containing a highconcentration of urea. In the absence of urea, these proteins migrate asa thick band. The 300 and 140 Kd proteins are only detectable ininfected cells, whereas the 125 Kd protein is detectable both ininfected cells as well as in the virus (FIG. 1A).

[0051] B. Identification of HIV-2 glycoproteins (FIG. 1B)

[0052] The glycosylation of the 300, 140, and 125 Kd proteins wasdemonstrated by metabolic labeling with ³H-glucosamine (FIG. 1B). Moreparticularly, HIV-2 CEM cells and T-lymphocytes were labeled with³H-glucosamine (200 μCi/ml; 4× 10⁶ cells/ml) for 18 hours. Extracts frominfected cells (lanes C) and culture medium containing virus (lanes V)were purified on the HIV-2 serum-Sepharose column. The labeled proteinswere analyzed by electrophoresis in a 7.5% gel. The lane on the far leftdepicts the result of electrophoresis in a 12.5% acrylamide gel andshows the presence of gp36. The gp36 is only slightly glycosylated andits detection required longer exposure times. Specifically, this part ofthe Figure had to be overexposed to see gp36; for this reasongp140/gp125 are resolved as a thick band.

[0053] The presence of gp300 and gp140 is not restricted to infected CEMcells. They are also detectable in HIV-2 infected T4 lymphocytes asdepicted in FIG. 1B. As in CEM cell cultures, gp300 and gp140 aredetectable only in infected cells, whereas gp125 is present both incells and in HIV-2 particles.

[0054] These results indicate that among the glycoproteins detectable inHIV-2 infected cells, gp125 and gp36 correspond to the virion envelope,whereas gp300 and gp140 might be precursors of the envelopeglycoproteins.

[0055] C. Characterization of gp300 and gp140 (FIG. 2)

[0056] The proteins gp300, gp140, and gp125 were labeled with³⁵S-methionine and analyzed by two dimensional gel electrophoresis. Thepatterns of resolution that were obtained indicated that gp300 and gp140are closely related.

[0057] More particularly, ³⁵S-methionine labeled gp300, gp140, and gp125purified from HIV-2 infected CEM cells (CELL) and culture mediumcontaining virus (SN) prepared in the same manner as the experimentsreported in FIG. 1 were analyzed by two dimensional gel electrophoresis(See “Experimental Procedures”). The pH gradient obtained by isoelectricfocusing (first dimension) is shown in FIG. 2. In the second dimension,proteins were resolved on a 7.5% polyacrylamide-SDS gel containing 6 Murea. Fluorographs of the gels are presented in FIG. 2.

[0058] Both proteins were resolved as an heterogeneous subspecies withidentical isoelectric points (pI) in the pH range of 6.8 to 7.8 (FIG.2). This similarity between gp140 nd gp300 suggested that gp300 is adimeric form of gp140 (see below).

[0059] The gp125, which is present in both infected cells and in virusparticles, exhibited less heterogeneity and migrated with pI valuesbetween 6.2 to 6.5. In infected cells, there was a minor subspecies ofgp125 with a pI value of 7.2 to 7.3. This basic gp125 is notincorporated into the HIV-2 virion. Thus it might represent aglycoprotein that is not processed properly. The acidic nature of themature gp125 might be due to the addition of sialic acid on some of itscarbohydrate side chains during the processing of the envelopeglycoprotein.

[0060] D. Dissociation of the native (a) and the denatured (b and c)gp300 (FIG. 3)

[0061] The gp300 is very stable since it resists ionic (1% SDS) andnon-ionic (2% Triton X-100) detergents, urea (2-6 M), high salt (1 MNaCl), and reducing agents (1% β-mercaptoethanol). However, it waspossible to demonstrate that gp300 could be dissociated into gp140 inacidic pH. In these experiments, immunoaffinity column bound proteinswere incubated in acetate buffer at pH values varying between 4 to 7.These samples were then analyzed by polyacrylamide gel electrophoresis.Fluorographs of the gels are shown in (a), (b), and (c) in FIG. 3. Insection (c), the band of gp300 and the dissociated gp140 were quantifiedby densitometric scanning of the fluorograph. More particularly, thegels were prepared as follows.

[0062] (a) ³⁵S-methionine labeled extracts from HIV-2 infected CEM cellswere purified on the HIV-2 serum-Sepharose column. This sample was thendivided into two equal aliquots: one was incubated in the bindingbuffer, FIG. 3(a), lane 1, whereas the other one was incubated in buffercontaining 30 mM sodium acetate pH 4.0, 0.2 mM PMSF, 100 units/mlaprotinin and 5 mM β-mercaptoethanol, FIG. 3(a), lane 2. After 1 hour at37° C., the acidic medium was neutralized and both samples were analyzedby electrophoresis.

[0063] (b) Purified and lyophilized ³⁵S-methionine labeled gp300 wassuspended in 100 μl of the sodium acetate buffer pH 4.0 as above FIG.3(b), lane 2. Incubations were carried out for 30 minutes at 37° C.before addition of 2-fold electrophoresis sample buffer containing 2 Murea. In lane 1 of FIG. 3(b), the lyophilized gp300 was directlysuspended in the electrophoresis sample buffer.

[0064] (c) The purified and lyophilized ³⁵S-methionine labeled gp300 wassuspended in solution containing 30 mM Tris-HCl, 0.2 mM PMSF and 100units/ml aprotinin and buffered with HCl at pH 7.5, 7.0, 6.5 and 6.0 (asindicated). After 60 minutes at 37° C., two fold electrophoresis samplebuffer was added and the samples were analyzed by electrophoresis.

[0065]FIG. 3(a) shows that the band of gp300 shifted to the position ofgp140 when the sample was incubated at pH 4. Further experiments werecarried out using purified preparations of gp300 obtained by preparativegel electrophoresis. Such denatured samples of gp300 were dissociatedcompletely in acetate buffer at pH 6.0, FIG. 3(b). The efficiency ofdissociation of the purified gp300 was probably due to a decrease in thepH along with the presence of residual SDS in the lyophilized sample,since column-bound native gp300 did not dissociate in the same buffer atpH values higher than pH 5 (data not shown).

[0066] In Tris-HCl buffer, the dissociation was less efficient. At pH7.5 there was only a slight dissociation of gp300 to gp140, but itincreased with decreasing pH values. In Tris buffer at pH 6.0, thedissociation was about 80%, (FIG. 3(c)). During dissociation of the puregp300 in either acetate or Tris-HCl buffer no proteins other than gp140were detectable (experiments carried out in 15% polyacrylamide gels;data not shown).

[0067] These results indicate that gp300 is a dimeric form of gp140, theprecursor of HIV-2 envelope glycoprotein. Thus, it seems most likelythat during the processing of the envelope glycoprotein, two moleculesof gp140 become fused by a pH-dependent mechanism.

[0068] E. Characterization of the Oligosaccharide Side Chains of HIV-2Glycoproteins (FIGS. 4 & 5)

[0069] Digestion with endo β-N-acetylglucosaminidase H (endo H)demonstrated the presence of N-linked oligosaccharides of the highmannose type on HIV-2 glycoproteins. The gp300, the gp140+ gp125, andthe gp125 were purified by immunoaffinity chromatography and preparativeelectrophoresis. (See “Experimental Procedures”). The lyophilizedsamples were suspended in endo H digestion buffer, which does notpromote the dissociation of gp300 to gp140. The procedure was carriedout as follows.

[0070] Purified and lyophilized gp300, gp140/gp125, and gp125(“Experimental Procedures”) were suspended in buffer containing 150 mMsodium citrate pH 5.5, 0.1% SDS (w/v), 0.5 mM PMSF before heating for 2minutes at 90° C. Aliquots of these samples were then incubated (2hours, 30° C.) without (lane 1, FIG. 4), or with 0.4 milli-units ofendo-H (lane 2, FIG. 4), 2 milli-units of endo-H (lane 3, FIG. 4), and10 milli-units of endo-H (lane 4, FIG. 4). All the reactions werestopped by the addition of two fold electrophoresis sample buffer.Electrophoresis was as previously described in relation to FIG. 1.Fluorographs of the different gels are shown in FIG. 4. The arrows p90and p80 on the right indicate the position of the digested product.Conditions for endo-H digestion were as described (Tarentino et al.,1974).

[0071] Upon endo H digestion, the electrophoretic mobility of gp300 wasreduced to a protein of 200-250 Kd. A small fraction of gp300 that hadbecome dissociated into gp140, was digested to give rise to a 80 Kdprotein (FIG. 4, section gp300, lane 4).

[0072] The gp140+gp125 sample was digested by endo H into 90 and 80 Kdproteins whereas gp125 was converted into a 90 Kd protein (FIG. 4,sections gp140/125 and gp125). These results indicate that endo Hdigestion of gp140 and gp125 give products of molecular weight 80 and 90Kd, respectively. The resistance to endo H digestion of gp125 relativeto gp140 is probably due to the conversion of some high mannose typeoligosaccharide side chains into complex oligosaccharides-duringprocessing of the envelope glycoprotein (Kornfeld and Kornfeld, 1985).

[0073] Metabolic labeling of cells was carried out with ¹⁴C-mannose and³H-fucose. More particularly, HIV-2 infected CEM cells were labeled (18hours) with ¹⁴C-mannose 25 μCi/ml; 4× 10⁶ cells/ml) or ³H-fucose (200μCi/ml; 4×10⁶ cells/ml). Extracts from infected cells (lanes C, FIG. 5)and culture medium containing virus (lanes V, FIG. 5) were purified onHIV-2 serum-Sepharose. Labeled glycoproteins were then analyzed bypolyacrylamide gel electrophoresis. A fluorograph is shown in FIG. 5.

[0074] Referring to FIG. 5, it will be apparent that metabolic labelingresulted in the incorporation of mannose into gp300, gp140, and gp125whereas only gp300 and gp125 were able to incorporate fucose. Fucoseresidues are normally transferred on oligosaccharide chains late in theglycosylation cycle, after the action of trimming enzymes of theendoplasmic reticulum and Golgi apparatus (Kornfeld and Kornfeld, 1985;Fuhrmann et al., 1985). The fact that gp140 does not contain fucoseresidues was consistent with it being the precursor of gp300 and gp125.

[0075] F. The Effect of Glycosylation Inhibitor Tunicamycin on theProcessing of HIV-2 Glycoproteins (FIG. 6)

[0076] All glycoproteins carrying N-linked glycans derive theiroligosaccharide moiety from the lipid-linked oligosaccharide,Glc₃Man_(q)-GlcNAc₂-pp-Dolichol, through a reaction carried out byprotein-oligosaccharidyl transferase, which catalyzes the en bloctransfer of oligosaccharide chains to asparagine residues (forreferences, see Kornfeld and Kornfeld, 1985). Tunicamycin blocks suchN-linked glycosylation since it inhibits the production ofN-acetylglucosamine pyrophosphoryldolichol, the first step in theassembly of lipid-linked oligosaccharides (Li et al, 1978; Heifetz etal., 1979).

[0077] In the presence of 2 μg/ml tunicamycin, the overall N-linkedglycosylation of HIV-2 envelope glycoproteins was completely blocked ininfected CEM cells. This was demonstrated by the lack of ³H-glucosamineincorporation in viral glycoproteins, gp300, gp140, and gp125.Inhibition of N-linked glycosylation by tunicamycin was carried out asfollows.

[0078] HIV-2 infected cells in the absence (lanes C, FIG. 6) or presence(TM, FIG. 6) of tunicamycin (2 μg/ml) were labeled with ³⁵S-methionine(panel “³⁵S-met”; 200 μCi/ml; 4×10⁶ cells/ml) or with ³H-glucosamine(panel “³H-GLcNAc”; 200 μCi/ml; 4×10⁶ cells/ml) for 16 hours. Cellstreated with tunicamycin were first incubated (37° C.) with theantibiotic (2 μg/ml) for 2 hours before labeling with ³⁵S-methionine or³H-glucosamine. Extracts from infected cells (CELL) and from the culturemedium containing virus (SN) were purified by HIV-2 serum-Sepharose andanalyzed by polyacrylamide 7.5% gel electrophoresis. Fluorographs of thegels are presented in FIG. 6. The position of the unglycosylatedenvelope precursor (p90/80) and the unglycosylated dimer (200 Kd) areindicated by the small arrows on the right. These 90/80 Kd and 200 Kdproteins do not incorporate ³H-glucosamine (panel ³H-GlcNAc, cell laneTM).

[0079] Under these experimental conditions, protein synthesis was notaffected in infected cells treated with tunicamycin (data not shown).Such cultures isotopically labeled with ³⁵S-methionine accumulated twomajor proteins of apparent sizes 200 and 80-90 Kd, which migrated aswide bands (FIG. 6). The molecular weights of these proteins coincidewell with endo H digestion products of gp300, gp140, and gp125 (FIG. 4),thus suggesting that the 200 and 80-90 Kd proteins correspond tounglycosylated forms of HIV-2 envelope glycoproteins. The molecularweight of the 80-90 Kd protein corresponds to the expected molecularweight of unglycosylated HIV-2 envelope precursor estimated from itsnucleic acid sequence (Guyader et al., 1987). The 200 Kd protein isprobably the dimeric form of the unglycosylated envelope precursor.These results confirm that HIV-2 envelope proteins have N-linkedpolysaccharide chains.

[0080] Besides inhibition of glycosylation, tunicamycin treatmentinhibits the processing and export of the envelope glycoprotein sincethe 80-90 Kd protein was not found in the extracellular medium (FIG. 6,lanes SN). Oligosaccharide chains of HIV-2 envelope proteins, therefore,are probably involved in the cellular transport through the Golgiapparatus. The absence of unglycosylated forms of the envelope proteinin the extracellular medium of tunicamycin treated cells might also bedue to its rapid degradation. Several reports have suggested that theunglycosylated form of a protein is more sensitive to proteases than itsglycosylated form (Olden et al., 1978; Schwartz et al., 1976).Accordingly, the small molecular weight proteins in ³⁵S-methioninelabeled cells cultured with tunicamycin might represent partiallydegraded products of the unglycosylated envelope protein (FIG. 6).

[0081] G. Effect of Oligosaccharide Trimming Inhibitors on the Synthesisof HIV-2 Glycoproteins (FIG. 7)

[0082] Asparagine-linked oligosaccharides (Glc₃Man_(q)GlcNAc₂) ofglycoproteins undergo extensive modifications or processing followingtheir attachment to nascent proteins (reviewed by Kornfeld and Kornfeld,1985). The trimming reactions occur in the lumen of the roughendoplasmic reticulum (RER) and in the Golgi apparatus by specificglucosidases and mannosidases.

[0083] Processing of oligosaccharide chains of glycoproteins can bemanipulated with the aid of specific inhibitors of the trimmingglucosidases and mannosidases (reviewed by Schwarz and Datema, 1984;Fuhrmann et al., 1985). In these experiments, different trimminginhibitors were used to investigate the localization of HIV-2glycoprotein precursors and also to study the role of glycosylation inthe processing of the envelope precursor. The inhibitors used were:

[0084] castanospermine, a plant alkaloid that inhibits glucosidase I(Saul et al., 1983);

[0085] deoxynojirimycin (dNM), a glucose analogue that inhibits trimmingglucosidase I and II (Lemansky et al., 1984);

[0086] 1-deoxymannojirimycin (dMM), a mannose analogue that inhibitsmannosidase catalyzed reactions (Fuhrmann et al., 1984);

[0087] bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene) thatinhibits glucosidase II (Datema et al., 1982); and

[0088] swainsonine, an indolizidine alkaloid that inhibits Golgimannosidase II (Tulsiani et al., 1982).

[0089] Specifically, HIV-2 infected CEM cells were labeled (16 hours,37° C.) with ³⁵S-methionine (200 μCi/ml; 4×10⁶ cells/ml) in the absence(lanes T. FIG. 7) or presence of the oligosaccharide trimming inhibitor

[0090] 1 mM bromoconduritol (lanes Bro, FIG. 7);

[0091] 1 mM castanospermine (lanes Cast, FIG. 7);

[0092] 10 μg/ml swainsonine (lanes Sw, FIG. 7);

[0093] 3 mM deoxynojirimycin (lanes dNM, FIG. 7); and

[0094] 1 mM deoxymannojirimycin (lanes dMM, FIG. 7).

[0095] Extracts from infected cells (panel CELL) and from culture mediumcontaining virus particles (panel SN) were purified on HIV-2serum-Sepharose to identify viral glycoproteins gp125, gp140, and gp300in infected cells and gp125 in culture medium. All samples were analyzedby polyacrylamide (7.5%) gel electrophoresis.

[0096] In order to show that inhibition of gp125 production by cellstreated with different inhibitors is specific to the viral glycoprotein,culture media were assayed for viral core protein p26 by animmunoprecipitation assay using an HIV-2 seropositive serum (Clavel etal., 1986a, 1987). The p26 was analyzed by polyacrylamide (12.5%) gelelectrophoresis. FIG. 7 represents a fluorograph showing only one partof each gel.

[0097] As expected, control infected cells contained gp300, gp140, andgp125 whereas only gp125 was observed in the extracellular medium (FIG.7, sections cell and SN, lanes T). In cells treated with castanospermineor dNM, there was a normal level of gp300, no gp125 and a small amountof a 150 Kd protein that probably corresponds to the glucosylated formof gp140. In such cells, therefore, the processing of the envelopeglycoprotein was blocked since no gp125 was detectable in theextracellular medium in spite of the production of p26, the core proteinof HIV-2 (FIG. 7, lanes Cast and dNM). These results indicate thatremoval of the terminal glucose residues from the oligosaccharide chainsof the envelope glycoprotein precursor is necessary for its processingand cleavage by the cellular protease.

[0098] Bromoconduritol, which acts on glucosidase II, also inhibited by70-90% the normal production of gp125, but the levels of gp140 and gp300remained normal (FIG. 7, lanes Bro). In contrast to castanospermine anddNM (which inhibit removal of terminal glucose residue), bromoconduritoltreatment (which inhibits removal of two inner glucose residues) did notblock completely the processing of HIV-2 envelope glycoprotein. In fact,low amounts of gp125 were detectable intracellularly andextracellularly. This latter result suggests that a low level of mannosetrimming can occur without removal of the two inner glucose residues.Such a phenomenon has been observed previously for the processing ofother viral glycoproteins during bromoconduritol treatment (Datema etal., 1982).

[0099] Mannosidase inhibitors, swainsonine and dMM, did not cause anapparent modification in the level of intracellular gp300, gp140, andgp125, but the level of extracellular gp125 was 50% less than that fromthe corresponding control cells (FIG. 7, lanes Sw and dMM). Thus,although the oligosaccharide chain was only deglucosylated, theglycoprotein precursor was proteolytically cleaved to yield a proteinsimilar to gp125 but with a higher content of mannose, which probablyaffected the cellular transport of gp125. The molecular weight of theextracellular glycoprotein produced in the presence of dMM was slightlyhigher than that produced in the absence of the inhibitor. This isprobably due to the higher content of mannose residues in theextracellular protein synthesized by dMM-treated cells (FIG. 7, sectionSN).

[0100] It should be emphasized that the effects of trimming enzymeinhibitors on the processing of HIV-2 envelope glycoprotein werespecific since the synthesis (data not shown) and the production ofHIV-2 p26 was not affected at all (FIG. 7, section SN).

[0101] Effect of Castanospermine and Monensin on the Processing of HIV-2Glycoproteins (FIG. 8)

[0102] To study the intracellular processing of HIV-2 glycoproteins,pulse-chase experiments were performed. The results are shown in FIG. 8.More particularly, the experiments were carried out as follows:

[0103] (a) Pulse-chase experiments were performed in HIV-2 infected CEMcells in the absence (Control) or presence of 1 mM castanospermine(Cast.). Control: infected cells were incubated-1 hour at 37° C. inmethionine-free medium before 15 minutes pulse labeling with³⁵S-methionine (200 μCi/ml; 4×10⁵ cells/ml; lane 1, FIG. 8a). Theradioactive label was then chased in culture medium containing 5 mM coldmethionine for 0.5, 1.5, and 3 hours (in lanes 2, 3, and 4,respectively, FIG. 8a). Cast.: HIV-2 infected CEM cells were incubated(1 hour, 37° C.) in methionine-free medium containing castanosperminebefore 30 minutes pulse labeling with ³⁵S-methionine (lane 1, FIG. 8a).These cells were then chased as above, but in the presence ofcastanospermine for 0.5, 1.5, and 3 hours (lanes 2, 3, and 4,respectively, FIG. 8a).

[0104] The gp140 was the first protein detectable 15 minutes after pulselabeling. During the chase, gp300 became detectable at 0.5 hours,whereas gp125 became detectable at 1.5-3 hours. The fact that gp300 wasobserved after synthesis of gp140 and the fact that gp125 was detectableonly after formation of gp300 (FIG. 8A, lanes 1-4), suggest thatdimerization is an intermediate step necessary for the oligosaccharideprocessing towards the mature glycoprotein, gp125. This suggestion wasconfirmed by the use of castanospermine, which inhibits the trimming ofthe external glucose residue of polysaccharide chains.

[0105] After 30 minutes of pulse labeling in the presence ofcastanospermine, a 150 Kd protein was detectable along with gp300 (FIG.8, Cast., lane 1). The 150 Kd protein should correspond to gp140; theslight increase in the molecular weight of the first precursor isascribed to the presence of glucose residues in its oligosaccharidechains. Thus, gp140 synthesized in HIV-2 infected cells represents theprecursor glycoprotein without its glucose residues. Accordingly, the150 Kd protein (gp150) represents the first immature glycoprotein ofHIV-2 envelope. The removal of glucose residues in control cells hasbeen reported to be a rapid process occurring during or briefly aftercotranslational translocation of precursor glycoproteins intoendoplasmic reticulum (Lemansly et al., 1984). After 30 minutes of pulseand 3 hours of chase in the presence of castanospermine, the level ofgp150 was gradually reduced while gp300 accumulated (FIG. 8, Cast, lanes1-4). Under these conditions, the precursor was not cleaved to yieldgp125.

[0106] Further characterization of HIV-2 envelope glycoprotein wasstudied in pulse-chase experiments using monensin, a cationic ionophorethat inhibits the transport of proteins from Golgi to the plasmamembrane or in some cases it might even block the transport of proteinsat the level of the medial Golgi cisternae (Tartakoff and Vassali, 1977;Johnson and Schlesinger, 1980; Strous and Lodish, 1980; Griffiths etal., 1983). HIV-2 infected cells in the absence or presence of monensinwere pulsed labeled as follows:

[0107] (b) Pulse chase experiments in HIV-2 infected cells were carriedout in the absence (Control) or presence of 1 μM monensin. Infectedcells with or without monensin were incubated (1 hour, 37° C.) inmethionine-free medium before 30 minutes pulse labeling with³⁵S-methionine (lanes 1, FIG. 8b). Labeled cells were then chased inculture medium containing 5 mM cold methionine for 0.5, 1.5, and 3 hours(lanes 2, 3, and 4, respectively, FIG. 8b). Extracts were purified onHIV-2 serum-Sepharose, and labeled proteins were analyzed bypolyacrylamide (7.5%) gel electrophoresis. Fluorographs are shown inFIG. 8b. (The p55 shows the gag precursor in section A, lanes 1.)

[0108] In the presence of monensin, HIV-2 infected cells synthesizednormal levels of gp140 and its dimeric form. However, no gp125 wasdetectable in monensin treated cells. After 1.5-3 hours of chase,monensin treated cells accumulated a 135 Kd protein (gp135) that isprobably the dissociated product of the dimer precursor. The slightlysmaller molecular weight of gp135 might be accounted for by the removalof some mannose residues by the action of RER and Golgi mannosidases. Inview of these results, it is tempting to speculate that afterdeglucosylation, gp300 becomes trimmed by mannosidases of RER and Golgibefore its dissociation into the mature precursor gp135 of HIV-2envelope. This gp135 could then be transported to plasma membrane andalso be cleaved by cellular protease. Inhibition of protein transport bymonensin blocks the mature glycoprotein gp135 in trans Golgi. No matureenvelope glycoproteins are detectable in monensin treated cells,intracellularly or extracellularly, although p26 is synthesized andexcreted (data not shown).

[0109] I. Dimerization of the Glycoprotein Precursor Occurs also inSIVmac Infected Cells (FIG. 9)

[0110] The nucleotide sequence of HIV-2 envelope shows a considerablehomology (75% amino acid identity) to that of SIV (Guyader et al., 1987;Chakrabarti et al., 1987; Franchini et al., 1987). For this reason, itwas important to investigate whether dimerization of envelopeglycoproteins is detectable in SIV infected cells. SIV proteins werepurified by the immunoaffinity column containing antibodies specific forHIV-2 proteins, since the gag, pol, and env proteins of HIV-2 and SIVare antigenically cross-reactive.

[0111] More particularly, SIV-infected HUT-78 cells were labeled (16hours, 37° C.) with ³⁵S-methionine (200 μCi/μl; 4×10⁶ cells/ml),³H-fucose (200 μCi/μl; 4×10⁶ cells/ml) and ¹⁴C-mannose (25 μCi/μl; 4×10⁶cells/ml). Extracts from infected cells (lanes C, FIG. 9) and from theculture medium containing SIV (lanes V, FIG. 9) were purified on HIV-2serum-Sepharose. Because of cross-reactivity between HIV-2 and SIVproteins, the HIV-2 positive serum could be used to immunoprecipitateSIV proteins. All samples were analyzed by polyacrylamide (7.5%) gelelectrophoresis. (See “Experimental Procedures”.) A fluorograph of thedifferent gels is shown in FIG. 9.

[0112]FIG. 9 shows that SIV infected cells synthesize three highmolecular weight proteins analogous to those synthesized in HIV-2infected cells: gp300, gp140, and gp130. The electrophoretic mobility ofgp300_(SIV) and gp140_(SIV) correspond to that of HIV-2 glycoproteinsgp300 and gp140 (data not shown). The gp130_(SIV) has a slightly highermobility than gp125 of HIV-2. The p55 labeled with ³⁵S-methionine isprobably the gag precursor of SIV.

[0113] Evidence that these proteins present in SIV infected cells areglycoproteins was provided by the isotopic labeling with ¹⁴C-mannose and³H-fucose. All the three proteins incorporated mannose, but onlygp300_(SIV) and gp130_(SIV) incorporated fucose (FIG. 9). Thegp300_(SIV) and gp140_(SIV) are intracellular proteins, whereasgp130_(SIV) is the extracellular glycoprotein. The fact that gp300_(SIV)and gp130_(SIV) can incorporate fucose suggests that they are processedproducts of gp140_(SIV).

[0114] These results indicate that doublet formation of the envelopeglycoprotein precursor is a specific property of HIV-2 and SIV envelopegene expression. It should be emphasized that HIV-1 envelopeglycoprotein does not undergo dimerization during its processing. HIV-1infected cells in the presence of castanospermine or dNM do notaccumulate envelope dimers (data not shown) as it is the case for HIV-2or SIV.

[0115] This invention thus describes for the first time the processingof HIV-2 envelope glycoproteins and details a novel mechanism ofglycoprotein processing with N-linked oligosaccharide chains. Theenvelope glycoproteins of HIV-2, i.e. the extracellular gp125 andtransmembrane gp36, arise from a common precursor glycoprotein (Guyaderet al., 1987). The unusual feature of this glycoprotein precursor isthat it requires the formation of a homologous dimer in order to becometransported and processed through the Golgi apparatus. The mechanism ofdimerization of envelope glycoprotein is not entirely clear. The factthat the purified dimer can be dissociated at an acidic pH (pH 6.0)suggests that dimerization might be pH dependent. Oligosaccharide chainson the precursor glycoprotein are not essential for dimer formation.Evidence for this has been obtained by two different experiments: (1)Digestion with endo H results in a shift in the electrophoretic mobilityof the dimer without dissociating it; and (2) In the presence oftunicamycin, HIV-2 infected cells synthesize an unglycosylated envelopeprecursor (80-90 Kd) that can form a dimer (200 Kd). These resultsemphasize that the dimer formation is an intrinsic property of thepolypeptide moiety of the envelope precursor.

[0116] Pulse-chase experiments in the absence or presence ofcastanospermine (FIG. 8) suggest that dimerization of the glycoproteinprecursor normally occurs immediately after removal of glucose residues.Since glucosidases are associated with membranes of endoplasmicreticulum, then it is most likely that dimerization occurs in the RER.In the presence of castanospermine, the dimer becomes accumulated in RERand it is not processed. However, once the glucose residues are removed,then inhibition of the RER mannosidase does not prevent the processingof the glycoprotein-dimer through the Golgi apparatus (FIG. 7).Accordingly, the glucose residues in the oligosaccharide chains of thedimer precursor prevent its exit from the RER. In accord with this, ithas been postulated that glucose trimming is necessary for efficienttransport from the RER to the Golgi, possibly because the deglucosylatedoligosaccharide forms part of a recognition site for a transportreceptor (Lodish and Kong, 1984; Lemansky et al., 1984). It might alsobe possible that glucose removal is crucial for the precursor dimer toachieve a correct functional configuration (Schlesinger et al., 1984)that favors the action of trimming mannosidases.

[0117] In view of these results, a schematic pathway for the processingof HIV-2 envelope glycoproteins is proposed in FIG. 10. With referenceto FIG. 10, the expected size of the polypeptide moiety of the precursorenvelope glycoprotein is about 80 Kd (FIGS. 4 and 6). Theoligosaccharide chain is transferred from dolichol-P-P to the newlysynthesized envelope precursor (80 Kd) probably at acceptor amino-acidasparagine residues (Kornfeld and Kornfeld, 1985). As depicted in FIG.10, tunicamycin inhibits assembly of dolichol-P-P glycan, and for thisreason the 80 Kd protein does not become glycosylated.

[0118] Addition of oligosaccharide chains to the 80 Kd protein yieldsthe first envelope glycoprotein precursor, gp150. This precursor mightor might not exist as such in infected cells, since addition ofpolysaccharide chains and glucose trimming probably occurs duringtranslation of the precursor. Whatever is the case, gp150 becomesrapidly deglucosylated to give gp140. At this stage, a difference inenvironment, perhaps of pH, would trigger dimer formation by the fusionof two gp140 molecules. The resulting gp300 can then be trimmed by theRER mannosidase and transported to the Golgi apparatus.

[0119] In the presence of castanospermine or dNM, gp150 becomesdimerized and is accumulated in the RER. This dimer is not processedbecause it is glucosylated. However, as long as the dimer is found inthe deglucosylated form, it can be transported to the Golgi; inhibitionof RER mannosidase by dMM does not block processing of the dimerprecursor.

[0120] In the Golgi, gp300 traverses the different compartments probablyby vesicular transport (Griffiths and Simons, 1986) during which theoligosaccharide chain is further trimmed by Golgi mannosidases beforeaddition of other sugars such as fucose and sialic acid. Evidence forfucose incorporation has been obtained by isotopic labeling of gp300with ³H-fucose. Evidence for sialic acid incorporation was obtainedindirectly by digesting gp300 with neuraminidase, an enzyme thathydrolyzes terminal N-acetylneuraminic acid in various glycoproteins(Peyrieras et al., 1983). The gp300 of HIV-2 is susceptible to digestionwith neuraminidase as evidenced by a significant decrease in theelectrophoretic mobility of the dimer (data not shown). The results areconsistent with the precursor keeping its dimeric form all through itsprocessing in the Golgi cis, medial, and trans cisternae before itstransport to the trans-Golgi network (TGN; Griffiths and Simons, 1986)where it dissociates due to a drop in the pH of this compartment.

[0121] The dissociated dimer yields glycoproteins (gp135) of slightlysmaller molecular weight than the first detectable glycoproteinprecursor (gp150-140). The gp135 could then be transported to plasmamembrane and also be cleaved by the cellular protease to yield themature glycoproteins of HIV-2 envelope, gp125, and gp36. Monensin mostprobably inhibits transport from the Golgi to the plasma membrane; forthis reason gp135 accumulates in the Golgi.

[0122] It is well accepted that the Golgi apparatus is implicated in themechanism of sorting secretory and plasma membrane proteins, which seemsto take place in the last Golgi compartment referred to as TGN(Griffiths and Simons, 1986). This compartment on the trans side of theGolgi stack, previously has been referred to as Golgi endoplasmicreticulum lysosomes (GERL) and recently as post-Golgi vacuoles or thetrans-most cisternae of the Golgi stack (Novikoff, 1976; Saraste andKuismanen, 1984; Orci et al., 1987). Interestingly, the pH of the TGNhas been considered to be mildly acidic, i.e., about pH 6 (Anderson andPathak, 1985; Griffiths and Simons, 1986). The acidic pH in the TGNcould then account for the dissociation of the processed dimer.

[0123] The results discussed here illustrate that the processing of theenvelope glycoproteins of HIV-2 is a multistep process involving thesynthesis of an immature precursor gp150-140, the intermediary dimerprecursor gp300 and finally the mature precursor gp135. Despite theirevolutionary relationship, HIV-1 and HIV-2 have found differentmechanisms for the processing of their envelope glycoproteins. Whetheror not these differences are involved in their pathogenesis is underinvestigation.

[0124] Following is a more detailed description of the experimentalprocedures used in this invention.

[0125] II. EXPERIMENTAL PROCEDURES

[0126] A. Materials

[0127] L-(³⁵S)Methionine (specific activity 1000 Ci/mmol, L-(6-³H)Fucose (specific activity: 45-70 Ci/mmol), D-(6-³H)Glucosamine (specificactivity: 20-40 Ci/mmol and D-(U-¹⁴C)Mannose (specific activity: 200-300mCi/mmol) were purchased from Amersham (Amersham, UK). Bromoconduritol,castanospermine, 1-deoxymannojirimycin (dMM), 1-deoxynojirimycin (dNM),swainsonine and tunicamycin were obtained from Boehringer-Mannheim(Mannheim, West Germany). Endo B-N-acetylglucosaminidase H was fromCalbiochem (San Diego, USA). Ampholines were purchased from Pharmacia(Uppsala, Sweden).

[0128] B. Virus and Cells

[0129] HIV-1_(BRU) isolate of the human immunodeficiency virus type 1(Montagnier et al., 1984), HIV-2_(ROD) isolate of the humanimmunodeficiency virus type 2 (Clavel et al., 1986a), and Simianimmunodeficiency virus, SIVmac₁₄₂ (Daniel et al., 1985), were used inthis study.

[0130] The different cell lines and human lymphocytes were cultured insuspension medium RPMI-1640 (GIBCO-BRL, Cergy-Pontoise, France)containing 10% (v/v) fetal calf serum; 2 μg/ml polybrene (Sigma) wasadded for HIV infected cell cultures. CEM clone 13 cells are derivedfrom the human lymphoid cell line CEM (ATCC-CCL119) and express the T4antigen to a high level. Five days after infection with HIV-1_(BRU) orHIV-2_(ROD) isolates, about 80-90% of the cells produce viral particlesand can be identified by a cytopathic effect corresponding tovacuolization of cells and appearance of small syncytia.

[0131] The HUT-78 cell line is another human T4 positive lymphoid cellline (Gadzudar et al., 1980) that is highly permissive for thereplication of SIVmac₁₄₂ (Daniel et al., 1985). Peripheral bloodlymphocytes from healthy blood donors were stimulated for three dayswith 0.2% (w/v) phytohemagglutinin fraction P (Difco, Detroit, USA) inRPMI-1640 medium supplemented with 10% fetal calf serum. Cells were thencultured in RPMI-1640 medium containing 10% (v/v) T cell growth factor(TCGF, Biotest). After infection with HIV-2, lymphocytes were culturedin presence of 10% (v/v) TCGF and 2 μg/ml Polybrene.

[0132] C. Metabolic Labeling of Cells

[0133] For metabolic labeling of proteins, infected cells were incubatedfor 16 hours at 37° C. in MEM culture medium without L-methionine andserum but supplemented with 200 μCi/ml ³⁵S-methionine. For metaboliclabeling of glycoproteins, infected cells were incubated for 16 hours at37° C. in MEM culture medium lacking serum and glucose but supplementedwith 200 μCi/ml ³H-fucose or 200 μCi/ml ³H-glucosamine or 25 μCi/ml¹⁴C-mannose.

[0134] D. Cell and Viral Extracts

[0135] Cell pellets corresponding to 10⁷ cells were resuspended in 100μl of buffer: 10 mM Tris-HCl pH 7.6, 15 mM NaCl, 1 mM EDTA, 0.2 mM PMSF,100 units/ml aprotinin (Iniprol, Choay) before addition of 100 μl of thesame buffer containing 2% (v/v) Triton X-100. Cell extracts werecentrifuged at 12,000 g for 10 minutes, and the supernatant was storedat −80° C. until used. For viral extract preparations, 100 μl of 10%lysis buffer (100 mM Tris-HCl pH 7.6, 1.5 M NaCl, 10 mM EDTA, 10% (v/v)Triton X-100, 100 units/ml aprotinin) was added per ml of clarifiedsupernatant from infected CEM cells and processed as above.

[0136] E. Preparation of an Inmunoadsorbant with Antibodies from anHIV-2 Seropositive Patient Sera

[0137] Immunoglobulins from the serum of an HIV-2 seropositive patientwere precipitated with 50% (NH₄)₂SO₄, dissolved in 20 mM sodiumphosphate (pH 8.0) and further purified on a DEAE cellulose column (DE52, Whatman) by elution with 20 mM sodium phosphate (pH 8.0).Immunoglobulins purified in this manner were judged to be 90% pure. Theantibodies were subsequently coupled to CNBr-activated Sepharose CL 4Baccording to a technique described by Berg (1977). Two milligrams of IgGwere coupled per ml of Sepharose CL 4B. This immunoadsorbant is referredto as HIV-2 serum-Sepharose.

[0138] F. Binding of the HIV-2 Proteins on the Immunoaffinity Column

[0139] Cell extracts from HIV-2 producing CEM cells were first dilutedin two volumes of binding buffer (20 mM Tris-HCl pH 7.6, 50 mM KCl, 150mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 20% (v/v) glycerol, 7 mMmercaptoethanol, 0.2 mM PMSF, 100 units/ml aprotinin) before incubationwith one volume of HIV-2 serum-Sepharose. Supernatants from HIV-2producing cells were processed as cell extracts except that only onetenth of binding buffer concentrate 10X was added per volume ofsupernatant. The binding was carried out overnight, then the column waswashed batchwise in binding buffer. Proteins bound to the column wereeluted by boiling in electrophoresis sample buffer (125 mM Tris-HCl pH6.8, 1% (w/v) SDS, 2 M urea, 20% glycerol, 1% β-mercaptoethanol). Elutedproteins were resolved by electrophoresis on 7.5% polyacrylamide-SDSgels containing 6 M urea and 0.1% bisacrylamide instead of 0.2% (w/v).

[0140] G. Preparative Electrophoresis

[0141] HIV-2 glycoproteins eluted from the affinity column were resolvedby polyacrylamide gel electrophoresis as previously described, and theregions of the gel containing the viral glycoproteins were cut out byreference to the position of prestained molecular weight protein markers(BRL).

[0142] Glycoproteins were eluted by incubation for 16 hours at 4° C. inelution buffer (0.1 M NaHCO₃, 0.5 mM EDTA, 0.05% (W/v) SDS, 0.2 mMPMSF). The glycoprotein fractions thus obtained were lyophilized andkept refrigerated until used.

[0143] H. Two Dimensional Electrophoresis

[0144] Two dimensional gel electrophoresis was performed as described byO'Farrel (1975) with the following modification: L-(³⁵S)-methioninelabeled proteins bound on the HIV-2 serum-Sepharose column were elutedby boiling in electrophoresis sample buffer as previously describedbefore dilution in a volume of buffer containing 9.5 M urea, 8% (v/v)mercaptoethanol, 1.6% (w/v) ampholines pH ranges 6.5-9, 0.4% (w/v)ampholines pH ranges 3-10, and 100 units/ml aprotinin.

[0145] It will be understood that the present invention is intended toencompass the previously described proteins and glycoproteins inpurified form, whether or not fully glycosylated, and whether obtainedusing the techniques described herein or other methods. In a preferredembodiment of this invention, the polypeptides are substantially free ofhuman tissue and human tissue components, nucleic acids, extraneousproteins and lipids, and adventitious microorganisms, such as bacteriaand viruses. It will also be understood that the invention encompassesequivalent proteins and glycoproteins having substantially the samebiological and immunogenic properties. Thus, this invention is intendedto cover serotypic variants of the proteins and glycoproteins of theinvention.

[0146] The proteins and glycoproteins of this invention can be obtainedby culturing HIV-2 in susceptible mammalian cells of lymphocyticlineage, such as T-lymphocytes or pre-T-lymphocytes of human origin ornon-human primate origin (e.g. chimpanzee, African green monkey, ormacaques.) A number of different lymphocytes expressing the CD4phenotypic marker can be employed. Examples of suitable target cells forHIV-2 infection are mononuclear cells prepared from peripheral blood,bone marrow, and other tissues from patients and donors. Alternatively,established cell lines can be employed. For example, HIV-2 can bepropagated on blood-donor lymphocyte cultures, followed by propagationon continuous cell strains of leukemic origin, such as HUT 78. HUT 78 isa well characterized mature human T cell line, which has been depositedat Collection Nationale Des Cultures De Micro- organismes (C.N.C.M.) atthe Institut Pasteur in Paris, France on Feb. 6, 1986, under culturecollection deposit accession number C.N.C.M. 1-519. Another suitabletarget for HIV-2 infection and production of the proteins andglycoproteins of the invention is the T-cell line derived from an adultwith lymphoid leukemia and termed HT. HT cells continuously producevirus after parental cells are repeatedly exposed to concentrated cellculture fluids harvested from short-term culture T-cells grown in TCGFthat originated from patients with LAS or AIDS. In addition, there areseveral other T or pre-T human cell lines, such as CEM and MOLT 3, thatcan be infected and continue to produce HIV-2. Furthermore,B-lymphoblastic cell lines can also be productively infected by HIV.Montagnier et al., Science, 225:63-66 (1984).

[0147] The proteins and glycoproteins of the invention can be producedin the target cells using the culture conditions previously described,as well as other standard techniques. For instance, infected humanlymphocytes can be stimulated for three days by phytohemaglutinin (PHA).The lymphocytes can be cultured in RPMI-1640 medium to which has beenadded 10% fetal calf serum, 10⁻⁵M beta-mercaptoethanol, interleukin-2,and human alpha anti-interferon serum. Barre-Sinoussi et al., Science,220:868-871 (1983). In addition, techniques for the propagation of HIV-2in HUT 78 and CEM cell lines are described in U.S. application Ser. No.835,228, filed Mar. 3, 1986, now U.S. Pat. No. 4,839,288, the entiredisclosure of which is relied upon and specifically incorporated byreference.

[0148] The production of virus in the cell cultures can be monitoredusing several different techniques. Supernatant fluids in the cellcultures can be monitored for viral reverse transcriptase activity.Electron microscopic observation of fixed and sectioned cells can alsobe used to detect virus. In addition, virus can be detected bytransmitting the virus to fresh normal human T-lymphocytes (e.g.,umbilical cord blood, adult peripheral blood, or bone marrow leukocytes)or to established T-cell lines. Testing for antigen expression byindirect immunofluorescence or Western Blot procedures using serum fromseropositive donors can also be employed. In addition, nucleic acidprobes can be utilized to detect viral production.

[0149] After a sufficient period of time for viral multiplication totake place, infected cells can be separated from the culture medium anddisrupted to expose intracellular proteins using conventionaltechniques. For example, physical shearing, homogenization, sonication,detergent solubilization, or freeze-thawing can be employed. The viralproteins released by these cells can be separated from the othercellular components and purified using standard biochemical procedures.For example, proteins can be separated from the live virus bycentrifugation, and the proteins can then be purified byultracentrifugation, gel filtration, ion-exchange chromatography,affinity chromatography, dialysis, or by the use of monoclonalantibodies or by combinations of these procedures. A thoroughpurification of the antigens of the invention can be performed byimmunoreaction with the sera of patients known to possess antibodieseffective against the antigens, with concentrated antibody preparationssuch as polyclonal antibodies, or with monoclonal antibodies directedagainst the antigens of the invention.

[0150] The proteins and the glycoproteins of the present invention canbe used as antigens to identify antibodies to HIV-2 and SIV in materialsand to determine the concentration of the antibodies in those materials.Thus, the antigens can be used for qualitative or quantitativedetermination of the retrovirus in a material. Such materials of courseinclude human tissue and human cells, as well as biological fluids, suchas human body fluids, including human sera. When used as a reagent in animmunoassay for determining the presence or concentration of theantibodies to HIV-2, the antigens of the present invention provide anassay that is convenient, rapid, sensitive, and specific.

[0151] More particularly, the antigens of the invention can be employedfor the detection of HIV-2 by means of immunoassays that are well knownfor use in detecting or quantifying humoral components in fluids. Thus,antigen-antibody interactions can be directly observed or determined bysecondary reactions, such as precipitation or agglutination. Inaddition, immunoelectrophoresis techniques can also be employed. Forexample, the classic combination of electrophoresis in agar followed byreaction with antiserum can be utilized, as well as i two-dimensionalelectrophoresis, rocket electrophoresis, and immunolabeling ofpolyacrylamide gel patterns (Western Blot or immunoblot.) Otherimmunoassays in which the antigens of the present invention can beemployed include, but are not limited to, radioimmunoassay, competitiveimmunoprecipitation assay, enzyme immunoassay, and immunofluorescenceassay. It will be understood that tubidimetric, calorimetric, andnephelometric techniques can be employed. An immunoassay based onWestern Blot technique is preferred.

[0152] Immunoassays can be carried out by immobilizing one of theimmunoreagents, either an antigen of the invention or the antibodies tothe antigen, on a carrier surface while retaining immunoreactivity ofthe reagent. The reciprocal immunoreagent can be unlabeled or labeled insuch a manner that immunoreactivity is also retained. These techniquesare especially suitable for use in enzyme immunoassays, such as enzymelinked immunosorbent assay (ELISA) and competitive inhibition enzymeimmunoassay (CIEIA).

[0153] When either the antigen of the invention or antibody to theantigen is attached to a solid support, the support is usually a glassor plastic material. Plastic materials molded in the form of plates,tubes, beads, or disks are preferred. Examples of suitable plasticmaterials are polystyrene and polyvinyl chloride. If the immunoreagentdoes not readily bind to the solid support, a carrier material can beinterposed between the reagent and the support. Examples of suitablecarrier materials are proteins, such as bovine serum albumin, orchemical reagents, such as glutaraldehyde or urea. Coating of the solidphase can be carried out using conventional techniques.

[0154] Depending on the use to be made of the proteins and glycoproteinsof the invention, it may be desirable to label them. Examples ofsuitable labels are radioactive labels, enzymatic labels, fluorescentlabels, chemiluminescent labels, and chromophores. The methods forlabeling proteins and glycoproteins of the invention do not differ inessence from those widely used for labeling immunoglobulin. The need tolabel may be avoided by using labeled antibody to the antigen of theinvention or anti-immunoglobulin to the antibodies to the antigen as anindirect marker.

[0155] Once the proteins and glycoproteins of the invention have beenobtained, they can be used to produce polyclonal and monoclonalantibodies reactive therewith. Thus, a protein or glycoprotein of theinvention can be used to immunize an animal host by techniques known inthe art. Such techniques usually involve inoculation, but they mayinvolve other modes of administration. A sufficient amount of theprotein or the glycoprotein is administered to create an immunogenicresponse in the animal host. Any host that produces antibodies to theantigen of the invention can be used. Once the animal has been immunizedand sufficient time has passed for it to begin producing antibodies tothe antigen, polyclonal antibodies can be recovered. The general methodcomprises removing blood from the animal and separating the serum fromthe blood. The serum, which contains antibodies to the antigen, can beused as an antiserum to the antigen. Alternatively, the antibodies canbe recovered from the serum. Affinity purification is a preferredtechnique for recovering purified polyclonal antibodies to the antigenfrom the serum.

[0156] Monoclonal antibodies to the antigens of the invention can alsobe prepared. One method for producing monoclonal antibodies reactivewith the antigens comprises the steps of immunizing a host with theantigen; recovering antibody-producing cells from the spleen of thehost; fusing the antibody-producing cells with myeloma cells deficientin the enzyme hypoxanthine-guanine phosphoribosyl transferase to formhybridomas; selecting at least one of the hybridomas by growth in amedium comprising hypoxanthine, aminopterin, and thymidine; identifyingat least one of the hybridomas that produces an antibody to the antigen;culturing the identified hybridoma to produce antibody in a recoverablequantity; and recovering the antibodies produced by the culturedhybridoma.

[0157] These polyclonal or monoclonal antibodies can be used in avariety of applications. Among these is the neutralization ofcorresponding proteins. They can also be used to detect viral antigensin biological preparations or in purifying corresponding proteins,glycoproteins, or mixtures thereof, for example when used in affinitychromatographic columns.

[0158] The invention provides immunogenic proteins and glycoproteins,and more particularly, protective polypeptides for use in thepreparation of vaccine compositions against HIV-2. These polypeptidescan thus be employed as viral vaccines by administering the polypeptidesto a mammal susceptible to HIV-2 infection. Conventional modes ofadministration can be employed. For example, administration can becarried out by oral, respiratory, or parenteral routes. Intradermal,subcutaneous, and intramuscular routes of administration are preferredwhen the vaccine is administered parenterally.

[0159] The ability of the proteins, glycoproteins, and vaccines of theinvention to induce protective levels of neutralizing antibody in a hostcan be enhanced by emulsification with an adjuvant, incorporation in aliposome, coupling to a suitable carrier, or by combinations of thesetechniques. For example, the proteins and glycoproteins of the inventioncan be administered with a conventional adjuvant, such as aluminumphosphate and aluminum hydroxide gel, in an amount sufficient topotentiate humoral or cell-mediated immune response in the host.Similarly, the polypeptides can be bound to lipid membranes orincorporated in lipid membranes to form liposomes. The use ofnonpyrogenic lipids free of nucleic acids and other extraneous mattercan be employed for this purpose.

[0160] The immunization schedule will depend upon several factors, suchas the susceptibility of the host to infection and the age of the host.A single dose of the vaccine of the invention can be administered to thehost or a primary course of immunization can be followed in whichseveral doses at intervals of time are administered. Subsequent dosesused as boosters can be administered as needed following the primarycourse.

[0161] The proteins and vaccines of the invention can be administered tothe host in an amount sufficient to prevent or inhibit HIV-2 infectionor replication in vivo. In any event, the amount administered should beat least sufficient to protect the host against substantialimmunosuppression, even though HIV infection may not be entirelyprevented. An immunogenic response can be obtained by administering theproteins or glycoproteins of the invention to the host in an amount ofabout 10 to about 500 micrograms antigen per kilogram of body weight,preferably about 50 to about 100 micrograms antigen per kilogram of bodyweight. The proteins and vaccines of the invention can be administeredtogether with a physiologically acceptable carrier. For example, adiluent, such as water or a saline solution, can be employed.

[0162] In summary, proteins and glycoproteins, which are precursors ofHIV-2 and SIV envelope protein, have now been identified. In addition toproviding useful tools for detection of antibodies to the retrovirus inhumans and for raising neutralizing antibodies to HIV-2 in vitro and invivo, this invention adds to the base of knowledge relating toimmunodeficiency active proteins and glycoproteins of the AIDS viruses.The reference molecular weight was in the form of the following dyemarkers marketed by BRL Co.:

[0163] myosine 200 Kd

[0164] phosphorylase B 92.7 Kd

[0165] BSA 68 Kd

[0166] ovalbumin 43 Kd

[0167] alpha chymotrypsin 25.7 Kd

[0168] beta lactoglobulin 18.4 Kd

[0169] lysozyme 14.3 Kd.

[0170] Molecular weights were estimated within an accuracy of about±100%.

REFERENCES

[0171] Anderson, R. G. W. and Pathat, R. K., “Vesicles and Cisternae inthe Trans Golgi Apparatus of Human Fibroblasts are Acidic Compartments,”Cell, 40, 635-643 (1985).

[0172] Barré-Sinoussi, F. Chermann, J. C., Rey, F., Nugeyre, M. T.,Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vézinet-Brun, F.,Rouzioux, C., Rozembaum, W., and Montagnier, L., “Isolation of aT-lymphotropic Retrovirus From a Patient at Risk for Acquired ImmuneDeficiency Syndrome (AIDS),” Science, 220, 868-871 (1983).

[0173] Berg, K., “Sequential Antibody Affinity Chromatography of HumanLeucocyte Interferon,” Scand. J. Immnunol., 6, 77-86 (1977).

[0174] Brun-Vézinet, F., Rey, M. A., Katlama, C., Girard, P. H., Roulot,D., Yeni, P., Lenoble, L., Clavel, F., Alizon, M., Gadelle, S., Madjar,J. J., and Harzic, M., “Lymphadenopathy-associatied virus type 2 in AIDSand AIDS-related Complex,” Lancet, i, 128-132 (1987).

[0175] Chakrabarti, L., Guyader, M., Alizon, M., Daniel, M. D.,Desrosiers, R. C., Thiollais, P., and Sonigo, P., “Sequence of SimianImmunodeficiency Virus from Macaque and its Relationship to Other Humanand Simian Retroviruses,” Nature, 328, 543-547 (1987).

[0176] Clavel, F., Guétard, D., Brun-Vézinet, F., Chamaret, S., Rey, M.A., Santos-Ferreira, M. O., Laurent, A. G., Dauguet, C., Katlama, C.,Rouzioux, C., Klatzmann, D., Champalimaud, J. L., and Montagnier, L.,“Isolation of a New Retrovirus from West African Patients with AIDS,”Science, 233, 343-346 (1986a).

[0177] Clavel, F., Guyader, M., Guétard, D., Sallé, M., Montagnier, L.,and Alizon, M., “Molecular Cloning and Polymorphism of the ImmuneDeficiency Virus Type 2,” Nature, 324, 691-694 (1986b).

[0178] Clavel, F., Mansinho, K., Charet, S., Guétard, D., Favier, V.,Nina, J., Santos-Ferreira, M. O., Champalimaud, J. L., and Montagnier,L., “Human Immunodeficiency Virus Type 2 Infection Associated with AIDSin West Africa,” N. Eng. J. Med., 316, 1180-1185 (1987).

[0179] Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D.H., Greaves, M. F., and Weiss, R. A., “The CD4 (T4) Antigen is anEssential Component of the Receptor for the AIDS Retrovirus,” Nature,312, 763-767 (1984).

[0180] Daniel, M. D., Letvin, N. L., King, N. W., Kannagi, M., Sehgal,P. K. Hunt, R. D., Kanki, P. J., Essex, M., and Desrosiers, R. C.,“Isolation of a T-cell Tropic HTLV-III-like Retrovirus from Macaques,”Science, 228, 1201-1204 (1985).

[0181] Datema, R., Romero, P. A., Legler, G., and Schwartz, R. T.,“Inhibition of Formation of Complex Oligosaccharides by the GlucosidaseInhibitor Bromoconduritol,” Proc. Natl. Acad. Sci. USA, 79, 6787-6791(1982).

[0182] Franchini, G., Gurgo, C., Guo, H- G., Gallo, R. C., Collalti, F.,Fargnoli, K. A., Hall, L. F., Wong-Staal, F., and Reitz Jr, M. S.,“Sequence of Simian Immunodeficiency Virus and its Relationship to theHuman Immunodeficiency Viruses,” Nature, 328, 539-543 (1987).

[0183] Fuhrmann, U., Bause, E., Legler, G., and Ploegh, H., “NovelMonnosidase Inhibitor Blocking Conversion of High Mannose to ComplexOligosaccharides,” Nature, 307, 755-758 (1984).

[0184] Fuhrmann, U., Bause, E., and Ploegh, H., “Inhibitors ofoligosaccharide Processing,” Biochimica et Biorhysica Acta, 825, 95-110(1985).

[0185] Fultz, P. N., McClure, H. M., Anderson, D. C., Swenson, R. B.,Anand, R., and Srinivasan, A., “Isolation of a T-lymphotropic Retrovirusfrom Naturally Infected Sooty Mangabey Monkeys (Cercocebusatys),” Proc.Natl. Acad. Sci. USA, 83, 5286-5290 (1986).

[0186] Gallaher, W. R., “Detection of a Fusion Peptide Sequence in theTransmembrane Protein of Human Immunodeficiency Virus,” Cell, 50,327-328 (1986).

[0187] Gazdar, A. F., Carney, D. N., Bunn, P. A., Russel, E. K., Jaffe,E. S., Schechter, G. P., and Guccion, J. G., “Mitogen Requirements forthe in vitro Production of Cutaneous T-cell Lymphomas,” Blood, 55,409-417 (1980).

[0188] Griffiths, G., Quinn, P., and Warren G., “Dissection of GolgiComplex: Monensin Inhibits the Transport of Viral Membrane Proteins fromMedial to Trans Golgi Cisternae in Baby Hamster Kidney Cells Infectedwith Semliki Forest virus,” J. Cell. Biol., 96, 835-851 (1983).

[0189] Griffiths, G. and Simons, R., “The Trans Golgi Network: Sortingat the Exit Site of the Golgi Complex,” Science, 234, 438-443 (1986).

[0190] Guyader, M., Emerman, M., Sonigo, P., Clavel, F., Montagnier, L.,and Alizon, M., “Genome Organization and Transactivation of the HumanImmunodeficiency Virus Type 2,” Nature, 326, 662-669 (1987).

[0191] Heifetz, A., Keenan, R. W., and Elhein, A. D., “Mechanism ofAction of Tunicamycin on the UDP-GlcNAc: DolichylphosphateGlcNAc-1-phosphate Transferase,” Biochemistry, 18, 2186-2192 (1979).

[0192] Johnson, D. C. and Schlesinger M. J., “Vesicular Stomatitis Virusand Sindbis Virus Glycoprotein Transport to Cell Surface is Inhibited byIonophores,” Virology, 103, 407-424 (1980).

[0193] Kannagi, M., Yetz, J. M., and Letvin, N. L., “In vitro GrowthCharacteristics of Simian T-lymphotropic Virus Type III,” Proc. Natl.Acad. Sci. USA, 82, 7053-7057 (1985).

[0194] Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J. Guétard,D., Hercend, T., Gluckman, J. C., and Montagnier, L., “T. Lymphocyte T4Molecule Behaves as the Receptor for Human Retrovirus LAV,” Nature, 312,767-768 (1984).

[0195] Kornfeld, R. and Kornfeld, S., “Assembly of Asparagine-linkedOligosaccharides,” Ann. Rev. Biochem., 54, 631-664 (1985).

[0196] Kowaslski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, W. C.,Terwilliger, E., Dayton, A., Rosen, C., Haseltine, W., and Sodroski,“Functional Regions of the Envelope Glycoprotein of HumanImmunodeficiency Virus Type 1,” Science, 237, 1351-1355 (1987).

[0197] Krust, B., Laurent, A. G., Le Guern, A., Jeannequin, O.,Montagnier, L., and Hovanessian, A. G., “Characterization of aMonoclonal Antibody Specific for HIV-1 Precursor Glycoprotein,” AIDS, 2,17-24 (1988).

[0198] Lemansky, P., Gieselmann, V., Hasilik, A., and Von Fugura, K.,“Cathespsin D and β-hexosaminidase Synthesized in the Presence of1-deoxynojirimycin Accumulate in the Endoplasmic Reticulum,” J. Biol.Chem., 259, 10129-10135 (1984).

[0199] Levy, J. A., Hoffman, A. D., Kramer, S. M., Lanois, J. A.,Shimabukuro, J. M., and Oskiro, L. S., “Isolation of LymphocytopathicRetroviruses from San Francisco Patients with AIDS,” Science, 225,840-842 (1984).

[0200] Li, E., Tabas, I., and Kornfeld, S., “The Synthesis ofComplex-Type Oligosaccharides: Structure of the Lipid-linkedoligosaccharide of the Vesicular Stomatitis Virus G Protein,” J. Biol.Chem., 253, 7762-7770 (1978).

[0201] Lifson, J. D., Feinberg, M. B., Reyes, G. R., Rabin, L., BanapourB. Chakrabati, S., Moss, B., Wong-Staal, F., Steimer, K. S., andEngleman, E. G., “Induction of CD4-dependent Cell Fusion by theHTLV-III/LAV Envelope Glycoprotein,” Nature, 323, 725-728 (1986).

[0202] Lodish, H. F. and Kong, N., “Glucose Removal from N-linkedOligosaccharides is Required for Efficient Maturation of CertainSecretory Glycoproteins From the Rough Endoplasmic Reticulum to theGolgi Complex,” J. Cell. Biol., 98, 1720-1729 (1984).

[0203] Marsh, M. and Dalgleish, A., “How do Human ImmunodeficiencyViruses Enter Cells?” Immunology Today, 8, 369-371 (1988).

[0204] McClure, M. O., Marsh, M., and Weiss, R. A., “HumanImmunodeficiency Virus Infection of CD4-bearing Cells Occurs by apH-independent Mechanism,” EMBO J., 7, 513-518 (1988).

[0205] McDougal, J. S., Mawle, A., Cort, S. P., Nicholson, J. K. A.,Cross, G. D., Scheppler-Campbel, J. A., Hicks, D., and Sligh, J.,“Cellular Tropism of the Human Retrovirus HTLV-III/LAV: Role of T CellActivation and Expression of the T4 Antigen,” J. Immunol., 135,3151-3162 (1985).

[0206] McDougal, J. S., Kennedy, M. S., Sligh, J. M. Cort, S. P., Mawle,A., and Nicholson, J. K. A., “Binding of HTLV-III/LAV to T4+ Cells by aComplex of the 11OK Viral Protein and the T4 Molecule,” Science, 231,382-385 (1986).

[0207] Montagnier, L. and Alizon M., “The Human Immune Deficiency Virus(HIV): An Update,” Ann. Inst. Pasteur/Virol., 138, 3-11 (1987).

[0208] Montagnier, L., Chermann, J. C., Barré-Sinoussi, F., Chamaret, S.Gruest, J., Nugeyre, M. T., Rey, F., Dauguet, C., Axler-Blin, C.,Vézinet-Brun, F., Rouzioux, C., Saimot, A. G., Rozembaum, W., Gluckman,J. C., Klatzmann, D., Vilmer, E., Griscelli, C., Gazengel, C., andBrunet, J. B. (1984), “A New Human T-lymphotropic Retrovirus:Characterization and Possible Role in Lymphadenopathy and AcquiredImmune Deficiency Syndrome,” in Human T Cell Leukemia/Lymphoma Viruses,edited by Gallo, R. C., Essex, M. E., and Gross, L., Cold Spring HarborLaboratory, N.Y., pp. 363-379 (1984).

[0209] Montagnier, L., Clavel, F., Krust, B., Chamaret, S., Rey, F.,Barré-Sinoussi, F., and Chermann, J. C., “Identification andAntigenicity of the Major Envelope Glycoprotein of LymphadenopathyAssociated Virus,” Virology, 144, 283-289 (1985).

[0210] Novikoff, A. B., “The Endoplasmic Reticulum: A Cytochemist'sView,” Proc. Natl. Acad. Sci. USA, 73, 2781-2787 (1976).

[0211] O'Farrel, P. H., “High Resolution Two-dimensional Electrophoresisof Proteins,” J. Biol. Chem., 250, 4007-4021 (1975).

[0212] Olden, K., Pratt, R. M., and Yamada, K. M., “Role ofCarbohydrates in Protein Secretion and Turnover: Effects of Tunicamycinon the Major Cell Surface Glycoprotein of Chick Embryo Fibroblasts,”Cell, 13, 461-473 (1978).

[0213] Orci, L., Ravazzola, M., Amherdt, A. P., Powel, S. K., Quinn, D.L, and Moore, H- P. H., “The Trans-Most Cisternae of the Golgi Complex:A Compartment for Sorting of Secretory and Plasma Membrane Proteins,”Cell, 51, 1039-1051 (1987).

[0214] Peyrieras, N., Bause, E., Legler, G., Vasilof, R. Claesson, L.,Peterson, P., and Ploegh, H., “Effects of Glucosidase InhibitorsNojirimycin and Deoxynojirimycin on the Biosynthesis of Membrane andSecretory Glycoproteins,” EMBO J., 2, 823-832 (1983).

[0215] Sonigo, P., Alizon, M., Staskus, K., Klatzmann, D., Cole, S.,Danos, O., Retzel, E., Tiollais, P., Haase, A., and Wain-Hobson, S.,“Nucleotide Sequence of the Visna Lentivirus: Relationship to the AIDSVirus,” Cell, 42, 369-382 (1985).

[0216] Stein, B. S., Gowda, S. D., Lifson, J. D., Penhallow, R. C.,Bensch, K. G., and Engleman, E. G., “pH-independent HIV-entry intoCD4-Positive T Cells via Virus Envelope Fusion to Plasma Membrane,”Cell, 49, 659-668 (1987).

[0217] Strous, G. J. A. M. and Lodish, H. F., “Intracellular Transportof Secretory and Membrane Proteins in Hepatoma Cells Infected byVesicular Stomatitis Virus,” Cell, 22, 709-717 (1980).

[0218] Tarentino, A. L., Plummer, T. H., and Maley, F., “The Release ofIntact Oligosaccharides From Specific Glycoproteins By Endo-B-N-AcetylGlucosaminidase H,” J. Biol. Chem., 249, 818-824 (1974).

[0219] Tartakoff, A. M. and Vassalli, P., “Plasma Cell ImmunoglobulinSecretion: Arrest is Accompanied by Alterations of the Golgi Complex,”J. Exp. Med., 14, 1332-1345 (1977).

[0220] Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S., and Alizon, M.,“Nucleotide Sequence of the AIDS Virus, LAV,” Cell, 40, 9-17 (1985a).

[0221] Wain-Hobson, S., Alizon, M., and Montagnier, L., “Relationship ofAIDS to Other Retroviruses,” Nature, 313, 743 (1985b).

[0222] Weiss, R. A., “Receptor Molecule Block HIV,” Nature, 331, 15(1988).

What is claimed is:
 1. An isolated immune complex comprising a proteinand an antibody that binds with said protein, wherein the protein isselected from the group consisting of gp300 of HIV-2, p200 of HIV-2,p90/80 of HIV-2, and gp300_(SIV).
 2. The immune complex of claim 1 ,wherein the antibody, protein, or both the antibody and protein, arelabeled with an immunoassay label selected from the group consisting ofradioisotopes, enzymes, fluorescent labels, chemiluminescent labels, andchromophore labels.
 3. An isolated antibody which binds with a proteinselected from the group consisting of gp300 of HIV-2, p200 of HIV-2,p90/80 of HIV-2, and gp300_(SIV).
 4. The antibody of claim 3 , whereinthe antibody is labeled with an immunoassay label selected from thegroup consisting of radioisotopes, enzymes, fluorescent labels,chemiluminescent labels, and chromophore labels.
 5. An immunogeniccomposition comprising a pharmaceutically effective amount of one ormore proteins of human immunodeficiency virus type 2 (HIV-2) and apharmaceutically acceptable carrier, wherein said proteins are selectedfrom the group consisting of gp300, p200, and p90/80 of HIV-2.
 6. An invitro diagnostic method for detecting infection of cells by humanimmunodeficiency virus type 2 (HIV-2), comprising: a) providing acomposition comprising cells suspected of being infected with HIV-2; b)disrupting cells in the composition to expose intracellular proteins;and c) assaying the exposed intracellular proteins for the presence ofone or more proteins selected from the group consisting of gp300 ofHIV-2, p200 of HIV-2, p90/80 of HIV-2, and gp300_(SIV), wherein thepresence of said one or more proteins is indicative of the presence ofHIV-2.
 7. The method of claim 6 , wherein the assaying of exposedintracellular proteins is carried out by a method selected from thegroup consisting of electrophoresis of the proteins and immunoassay ofthe proteins with antibodies that are immunologically reactive withgp300 of HIV-2, p200 of HIV-2, p90/80 of HIV-2, or gp300_(SIV).
 8. Themethod of claim 7 , wherein the antibodies are labeled with animmunoassay label selected from the group consisting of radioisotopes,enzymes, fluorescent labels, chemiluminescent labels, and chromophorelabels.
 9. An in vitro method for detecting antigens of humanimmunodeficiency virus type 2 (HIV-2), comprising: a) providing acomposition suspected of containing antigens of HIV-2; and b) assayingthe composition for the presence of one or more proteins selected fromthe group consisting of gp300, p200, and p90/80 of HIV-2, wherein thepresence of said one or more proteins is indicative of the presence ofantigens of HIV-2.
 10. The method of claim 9 , wherein said assaying ofthe composition is carried out by a method selected from the groupconsisting of electrophoresis of said proteins and immunoassay withantibodies that are immunologically reactive with gp300, p200, or p90/80of HIV-2.
 11. The method of claim 10 , wherein the antibodies arelabeled with an immunoassay label selected from the group consisting ofradioisotopes, enzymes, fluorescent labels, chemiluminescent labels, andchromophore labels.
 12. An in vitro diagnostic method of distinguishingHIV-2 infection, or co-infection of HIV-1 and HIV-2, from HIV-1infection in cells comprising: a) providing an extract comprisingintracellular proteins of said cells; and b) assaying said extract forthe presence of one or more proteins selected from the group consistingof gp300 of HIV-2, p200 of HIV-2, p90/80 of HIV-2, and gp300_(SIV),wherein the presence of said one or more proteins is indicative of thepresence of HIV-2 infection or co-infection of HIV-1 and HIV-2.
 13. Themethod of claim 12 , wherein said assaying of the extract is carried outby a method selected from the group consisting of electrophoresis ofsaid proteins and immunoassay with antibodies that are immunologicallyreactive with gp300, p200, or p90/80 of HIV-2.
 14. The method of claim13 , wherein the antibodies are labeled with an immunoassay labelselected from the group consisting of radioisotopes, enzymes,fluorescent labels, chemiluminescent labels, and chromophore labels. 15.An in vitro diagnostic method for detecting the presence or absence ofantibodies which bind to a protein of HIV-2, comprising: a) contactingone or more proteins of HIV-2 selected from the group consisting ofp90/80, p200, and gp300 of HIV-2 with a biological fluid for a time andunder conditions sufficient for said proteins and antibodies in thebiological fluid to form a protein-antibody immune complex; and b)detecting the formation of the complex.
 16. The method of claim 15 ,wherein the detecting step further comprises measuring the formation ofsaid immune complex.
 17. The method of claim 15 , wherein said one ormore proteins are labeled with an immunoassay label selected from thegroup consisting of radioisotopes, enzymes, fluorescent labels,chemiluminescent labels, and chromophore labels.
 18. An in vitrodiagnostic kit for detecting the presence or absence of antibodies whichbind to a protein of HIV-2, comprising: a) one or more proteins of HIV-2selected from the group consisting of p90/80, p200, and gp300 of HIV-2;and b) means for detecting the formation of immune complex between saidproteins and said antibodies; wherein the proteins and the means arepresent in an amount sufficient to perform said detection.
 19. The kitof claim 18 , wherein the means for detecting the formation of theimmune complex is an assay selected from the group consisting ofradioimmunoassay, immunoenzymatic assay, and immunofluorescent assay.20. An in vitro method for detecting antibodies in a sample of humanbody fluid which specifically bind to antigenic sites of an antigen,comprising: a) contacting said antigen with antibodies from human bodyfluid for a time and under conditions sufficient to permit formation ofan antigen-antibody complex between said antigen and said antibodies;and b) detecting the formation of said antigen-antibody complex, whereinsaid antigen comprises a protein selected from the group consisting ofp90/80 of HIV-2, p200 of HIV-2, gp300 of HIV-2, and gp300_(SIV).